Molecular Dynamics Simulations of Molecular Dynamics Simulations of Compressional Metalloprotein DeformationCompressional Metalloprotein Deformation

Andrew HungAndrew Hung11, Jianwei Zhao, Jianwei Zhao22, Jason J. Davis, Jason J. Davis22, Mark S. P. Sansom, Mark S. P. Sansom11

11 Department of Biochemistry, University of Oxford, South Parks Road, Oxford, U.K. OX1 3QUDepartment of Biochemistry, University of Oxford, South Parks Road, Oxford, U.K. OX1 3QU22 Department of Chemistry, University of Oxford, South Parks Road, Oxford, U.K. OX1 3QUDepartment of Chemistry, University of Oxford, South Parks Road, Oxford, U.K. OX1 3QU

BackgroundBackgroundAzurin, a Cu metalloprotein, is a biological electron transfer Azurin, a Cu metalloprotein, is a biological electron transfer agent. An understanding of the process of electron transfer agent. An understanding of the process of electron transfer through this protein is of immense technological interest in the through this protein is of immense technological interest in the development of new molecular electronic devicesdevelopment of new molecular electronic devices11. Recently, . Recently, the conduction properties of azurin were studied by conducting the conduction properties of azurin were studied by conducting atomic force microscopy (C-AFM)atomic force microscopy (C-AFM)22. In this experiment, the . In this experiment, the protein is immobilised to the AFM tip, and tunneling currents protein is immobilised to the AFM tip, and tunneling currents through the protein were measured at a range of bias voltages through the protein were measured at a range of bias voltages while under various extents of applied compressive force.while under various extents of applied compressive force.

Computational DetailsComputational DetailsMolecular dynamics (MD) simulations were performed under Molecular dynamics (MD) simulations were performed under constant particle number, volume and temperature (NVT) constant particle number, volume and temperature (NVT) conditions using GROMACSconditions using GROMACS33 The GROMOS96 forcefield The GROMOS96 forcefield parameters were employed with time steps of 2 fs. Potential energy parameters were employed with time steps of 2 fs. Potential energy cut-off radii of 10 Å were used for van der Waals’ and electrostatic cut-off radii of 10 Å were used for van der Waals’ and electrostatic interactions. Bond lengths were constrained via the LINCS interactions. Bond lengths were constrained via the LINCS algorithm. The protein was coupled to a temperature bath at 300 K. algorithm. The protein was coupled to a temperature bath at 300 K. Energy minimisation and 100ps of equilibration were performed on Energy minimisation and 100ps of equilibration were performed on the protein at each compression distance, with a subsequent 100ps the protein at each compression distance, with a subsequent 100ps of simulation collected on which analyses were performed. of simulation collected on which analyses were performed. Analyses of MD trajectories were performed using the suite of Analyses of MD trajectories were performed using the suite of software included in the GROMACS software. Visualisation of software included in the GROMACS software. Visualisation of system geometries and evaluation of protein secondary structure system geometries and evaluation of protein secondary structure were performed using the program VMDwere performed using the program VMD44..

AFM Tip-Protein-Surface ModelAFM Tip-Protein-Surface Model3-dimensional periodic boundary conditions. Surface constructed from 3-dimensional periodic boundary conditions. Surface constructed from united-atom CHunited-atom CH44 molecules. Azurin obtained from PDB file 4AZU molecules. Azurin obtained from PDB file 4AZU55 , ,

with all non-protein molecules removed. Compression of the protein with all non-protein molecules removed. Compression of the protein achieved by stepwise reduction of the achieved by stepwise reduction of the zz-direction cell length. MD runs -direction cell length. MD runs performed at each tip-surface distance.performed at each tip-surface distance.

Preliminary ResultsPreliminary ResultsSecondary structural features were maintained from initial tip-surface Secondary structural features were maintained from initial tip-surface separation of 42 separation of 42 Å to ~25 Å to ~25 Å. Packing density increases with Å. Packing density increases with compression within this range. At lower separations, protein unfolding compression within this range. At lower separations, protein unfolding occurs, and density decreases with compression. Consistent with AFM occurs, and density decreases with compression. Consistent with AFM conduction experiments which showed decrease in conduction experiments which showed decrease in φφ00 with compression with compression

up to a certain critical point before reaching a constant, minimum value. up to a certain critical point before reaching a constant, minimum value.

Further WorkFurther WorkPreliminary results from the current MD simulations have shown that Preliminary results from the current MD simulations have shown that simulations can contribute to our understanding of protein-mediated simulations can contribute to our understanding of protein-mediated tunneling under compressive stress. Work in progress include studying tunneling under compressive stress. Work in progress include studying the effects of hydration waters, Cu coordination changes under the effects of hydration waters, Cu coordination changes under compression, and using a more realistic AFM tip and surface model.compression, and using a more realistic AFM tip and surface model.

ReferencesReferences11 R. Rinaldi R. Rinaldi et al.et al., , Adv. MatAdv. Mat. 14, p1453 (2002). 14, p1453 (2002)22 J. Zhao, J. J. Davis, J. Zhao, J. J. Davis, NanotechnologyNanotechnology 14(9), p1023 (2003) 14(9), p1023 (2003)33 D. van der Spoel D. van der Spoel et alet al., ., Gromacs User Manual version 3.1Gromacs User Manual version 3.1, Groningen, The , Groningen, The Netherlands, Internet : www.gromacs.org (2002)Netherlands, Internet : www.gromacs.org (2002)44 W. Humphrey W. Humphrey et alet al., ., J. Molec. GraphicsJ. Molec. Graphics 14(1), p33 (1996) 14(1), p33 (1996)55 H. Nar H. Nar et alet al., ., J. Mol. Biol.J. Mol. Biol. 218, p427 (1991) 218, p427 (1991)

Figure 3. Stepwise compression of azurin at (z) 42 Å, 27 Å and 17 ÅFigure 3. Stepwise compression of azurin at (z) 42 Å, 27 Å and 17 Å

Figure 4. Secondary structure (a) and protein atomic packing density (b) Figure 4. Secondary structure (a) and protein atomic packing density (b) as a function of tip-surface distance. Red = as a function of tip-surface distance. Red = αα-helix, yellow = -helix, yellow = ββ-strands-strands

Figure 1. Schematic of a typical C-AFM experimentFigure 1. Schematic of a typical C-AFM experiment22..

Current-bias voltage Current-bias voltage I(V)I(V) curves were acquired at curves were acquired at different values of applied different values of applied forceforce22 (differentiated by (differentiated by colour), as shown in colour), as shown in Figure 2. Fitting each Figure 2. Fitting each curve to a modified curve to a modified Simmons model, a curve Simmons model, a curve of tunneling barrier height of tunneling barrier height φφ00 vs. applied force was vs. applied force was

obtained. obtained. φφ00 was found to was found to

initially decrease initially decrease monotonously with force, monotonously with force, but becomes constant but becomes constant above above ~~30nN. 30nN.

Figure 2. Current with Figure 2. Current with respect to bias voltage respect to bias voltage II((VV) ) curves for varying degrees of curves for varying degrees of applied force. (black = lowest applied force. (black = lowest applied force, yellow = applied force, yellow = highest)highest)

Molecular simulations have been performed to determine a Molecular simulations have been performed to determine a possible mechanism for this behaviour, with particular focus possible mechanism for this behaviour, with particular focus on the structure and packing density (on the structure and packing density (ρρ) ) changes with changes with respect to compression, as respect to compression, as φφ00 is believed to be related to is believed to be related to ρρ..

Current WorkCurrent Work